EP1374681A1 - Procédé pour identifier des composés à action fongicide - Google Patents

Procédé pour identifier des composés à action fongicide Download PDF

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Publication number
EP1374681A1
EP1374681A1 EP03013024A EP03013024A EP1374681A1 EP 1374681 A1 EP1374681 A1 EP 1374681A1 EP 03013024 A EP03013024 A EP 03013024A EP 03013024 A EP03013024 A EP 03013024A EP 1374681 A1 EP1374681 A1 EP 1374681A1
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Prior art keywords
phosphomannomutase
activity
polypeptide
fungicides
phosphate
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EP03013024A
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German (de)
English (en)
Inventor
Martin Dr. Vaupel
Ralf Dr. Dunkel
Karl-Heinz Dr. Kuck
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Bayer CropScience AG
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Bayer CropScience AG
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Publication of EP1374681A1 publication Critical patent/EP1374681A1/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi

Definitions

  • the invention relates to a method for identifying fungicides which Use of fungal phosphomannomutase to identify fungicides, and the use of phosphomannomutase inhibitors as fungicides.
  • fungicides are often found in essential biosynthetic pathways searched.
  • the ideal fungicides continue to be those substances, the gene products inhibit which is of crucial importance in the expression of pathogenicity of a mushroom.
  • the object of the present invention was therefore to find a suitable new one Identify the target for potential fungicidal agents and make them accessible and to provide a method for identifying Allows modulators of this point of attack, which are then used as fungicides can be.
  • Phosphomannomutase (EC 5.4.2.8), also known as D-mannose 1,6-phosphomutase, catalyzes the isomerization of D-mannose 1-phosphate to D-mannose 6-phosphate ( Figure 1).
  • D-mannose is 1,6-bisphosphate or D-glucose 1,6-bisphosphate necessary as a cofactor ( Figure 2).
  • Via a phosphate group transfer from the cofactor to D-mannose 1-phosphate is formed as an intermediate D-mannose 1,6-bisphosphate and by subsequent cleavage of the phosphate group on the C1 atom gives the end product D-mannose 6-phosphate and the recycled cofactor (Oesterhelt et al., 1997).
  • the phosphomannomutase response is an essential step in the deployment activated sugar for N- and O-glycosylation of proteins or the synthesis of glycosylphosphoinositol (GPI) anchors (Burda & Aebi 1999; Herscovics & Orlean, 1993; Hibbs et al .; 1988; Kepes, 1994).
  • GPI glycosylphosphoinositol
  • Genes for the phosphomannomutase were cloned from various organisms, for example from Saccharomyces cerevisiae (Swissprot Accession No .: P07283).
  • Candida albicans (Swissprot Accession No .: P31353), Schizosaccharomyces pombe (Swissprot Accession No .: AB000703), Homo sapiens (Swissprot: Accession No .: 015305) mouse (Swissprot: Accession No .: 035621) or Arabidopsis thaliana (Swissprot Accession No .: 080840).
  • phosphomannomutase is also present in prokaryotes such as Pseudomonas aeruginosa (EMBL Accession No .: M60873), Azospirillum brasilense (Swissprot Accession No. P45632) or Campylobacter jejuni (EMBL Accession No.:AL139078)
  • sequence similarities are within the eukaryotic classes or significant within the bacterial class, whereas the sequence identity between representatives of the eukaryotic class and the bacterial class is significant.
  • the phosphomannomutase was e.g. isolated from yeast, partially cleaned and also partially characterized (Kepes and Schekman, 1988; Glaser et al, 1966; Glaser et al., 1959).
  • the object of the present invention was therefore also phosphomannomutases from mushrooms as well as making methods accessible in which inhibitors of the enzyme identified and tested for their fungicidal properties can be.
  • identity refers to the number of Sequence positions that are identical in an alignment. It is usually in percent the alignment length.
  • similarity sets the definition ahead of a similarity metric, that is, a measure of how similar you are for example want to accept a valine to a threonine or a leucine.
  • homologous proteins have emerged from a common one Predecessor sequence developed.
  • the term doesn't necessarily have anything to do with identity or To do similarity, except that homologous sequences are usually more similar (or have more identical positions in an alignment) than non-homologous ones Sequences.
  • complete phosphomannomutase describes the phosphomannomutase by the full coding region a transcription unit is encoded, starting with the ATG start codon and comprehensive all information-bearing exon areas of the in the organism of origin Present gene coding for phosphomannomutase, as well as for a correct termination of the transcription necessary signals.
  • biological activity of a phosphomannomutase refers to the ability of a polypeptide described above described reaction, i.e. the conversion of D-mannose 1-phosphate to D-mannose Catalyze 6-phosphate.
  • active fragment does not describe more complete nucleic acids coding for phosphomannomutase, but still encode polypeptides with the biological activity of a phosphomannomutase, and the one characteristic of phosphomannomutase as above can catalyze described. Such fragments are shorter than the ones above described complete, for the phosphomannomutase coding nucleic acids. In this case, both at the 3 'and / or 5' ends of the sequence Nucleic acids have been removed, but parts of the sequence can also be deleted, i.e. the biological activity of phosphomannomutase not significantly affect.
  • a lower one, or possibly one increased activity, but which still characterizes or uses the resulting phosphomannomutase fragment is considered sufficient understood in the sense of the expression used here.
  • the expression “active Fragment” can also affect the amino acid sequence of phosphomannomutase relate and then applies analogously to the above statements for such polypeptides that certain parts no longer compared to the complete sequence defined above included, but the biological activity of the enzyme is not critical is impaired.
  • the fragments can have different lengths.
  • phosphomannomutase inhibition test or “inhibition test” as used herein used refers to a method or test that allows the Inhibition of the enzymatic activity of a polypeptide with the activity of a Phosphomannomutase through one or more chemical compounds (Candidate compound (s)) to recognize, making the chemical compound as Inhibitor of phosphomannomutase can be identified.
  • gene as used herein is the term for one Section from the genome of a cell used for the synthesis of a polypeptide chain responsible for.
  • Pythium species such as, for example, Pythium ultimum, Phytophthora species, such as, for example, Phytophthora infestans, Pseudoperonospora species, such as, for example, Pseudoperonospora humuli or Pseudoperonospora cubensis, Plasmopara species, such as, for example, Plasmopara viticola, Bremia species, such as, for example, Bremia lactucae, Peronospor Species such as Peronospora pisi or P.
  • brassicae Erysiphe species such as Erysiphe graminis, Sphaerotheca species such as Sphaerotheca fuliginea, Podosphaera species such as Podosphaera leucotricha, Venturia species such as Venturia inaequalis and Pyrenophora species , such as, for example, Pyrenophora teres or P.
  • graminea (conidial form: Drechslera, Syn: Helminthosporium), Cochliobolus species, such as, for example, Cochliobolus sativus ( Conidial form : Drechslera, Syn: Helminthosporium), Uromyces species, such as, for example, Uromyces appinia species, Pu such as, for example, Puccinia recondita, Sclerotinia species n, such as Sclerotinia sclerotiorum, Tilletia species, such as Tilletia caries; Ustilago species, such as, for example, Ustilago nuda or Ustilago avenae, Pellicularia species, such as, for example, Pellicularia sasakii, Pyricularia species, such as, for example, Pyricularia oryzae, Fusarium species, such as, for example, Fusarium culmorum, Botrytis species, Sept
  • Magnaporthe grisea Cochliobulus heterostrophus, Nectria hematococcus and Phytophtora species.
  • Fungicidal active ingredients using the phosphomannomutase according to the invention can be found, but can also with phosphomannomutase from human pathogenic Fungus species interact, interacting with the different ones phosphomannomutases occurring in these fungi are not always equally strong have to be.
  • the present invention therefore also relates to the use of Phosphomannomutase inhibitors for the manufacture of treatment agents of diseases caused by human pathogenic fungi.
  • Fungicidal active ingredients which are found with the aid of a phosphomannomutase obtained from a specific fungus, here from S. cerevisiae, can therefore also interact with phosphomannomutase from other numerous fungal species, especially with phytopathogenic fungi, the interaction with the different ones occurring in these fungi Phosphomannomutases do not always have to be equally strong. Among other things, this explains the observed selectivity of the substances active on this enzyme.
  • heterologous promoter refers to a promoter that has different properties than the promoter that in The organism of origin controls the expression of the gene in question.
  • agonist refers to a molecule that accelerates or enhances the activity of phosphomannomutase.
  • antagonist refers to a molecule that slows down or prevents the activity of phosphomannomutase.
  • modulator as used herein provides the generic term Agonist or antagonist.
  • Modulators can be small organic chemical Molecules, peptides or antibodies that bind to the polypeptides according to the invention bind or affect their activity.
  • modulators can be small organic chemical molecules, peptides or antibodies attached to a molecule bind, which in turn binds to the polypeptides according to the invention, and thereby influencing their biological activity.
  • Modulators can be natural Represent substrates and ligands or structural or functional mimetics from that.
  • modulator as used herein is preferred is, however, those molecules that are not the natural substrates or Represent ligands.
  • phosphomannomutase is a target protein (a so-called "Target") can be fungicidally active substances.
  • Previously known inhibitors of Phosphomannomutase, which are substrate analogs have no known fungicidal activity.
  • the phosphomannomutase an enzyme that is particularly important for fungi and therefore in particular Is suitable as a target protein for the search for further and improved fungicidally active ingredients to be used.
  • the Phosphomannomutase can be a target or "target” for fungicidal agents can, the inhibition of phosphomannomutase so damage or The fungus dies.
  • the enzyme phosphomannomutase was further than one Detected polypeptide, which is used to identify modulators or inhibitors of its enzymatic activity can be used in appropriate test procedures, what not given for various theoretically interesting targets is.
  • a method was therefore developed that is suitable, the activity of the phosphomannomutase and optionally the Inhibition of this activity in the presence of a potential inhibitor of the To determine phosphomannomutase in a so-called inhibition test on this Manner inhibitors of the enzyme e.g. identified in HTS and UHTS procedures and to test their fungicidal properties.
  • a potential inhibitor of the To determine phosphomannomutase in a so-called inhibition test on this Manner inhibitors of the enzyme e.g. identified in HTS and UHTS procedures and to test their fungicidal properties.
  • As part of the present Invention has also been shown to inhibit phosphomannomutase Mushrooms can be used as fungicides.
  • the phosphomannomutase can also be inhibited in vivo by active substances and that a fungal organism treated with these active substances can be damaged and killed by treatment with these active substances.
  • the inhibitors of a fungal phosphomannomutase can thus be used as fungicides in crop protection or as antifungals in pharmaceutical indications.
  • the inhibition of the phosphomannomutase with a substance identified in a method according to the invention leads to the damage or death of the treated fungi in synthetic media or on the plant.
  • the enzyme phosphomannomutase can be obtained, for example, from fungi such as S. cerevisiae .
  • the gene can be expressed, for example, recombinantly in Escherichia coli and an enzyme preparation can be prepared from E. coli cells (Example 1).
  • the associated ORF was amplified from genomic DNA according to methods known to the person skilled in the art via gene-specific primers.
  • the corresponding DNA was cloned into the vector pGEX-4T-1 (Pharmacia Biotech, enables the introduction of an N-terminal GST tag).
  • the resulting plasmid pSec53 contains the complete coding sequence of sec53 in an N-terminal fusion with a GST tag from the vector.
  • the Sec53 fusion protein has a calculated mass of 53 kD (Example 1 and Figure 4).
  • the plasmid pSec53 was then used for the recombinant expression of Sec53 in E. coli BL21 (DE3) cells (Example 1).
  • polypeptides with the activity of a phosphomannomutase can also be obtained from other fungi, preferably from phytopathogenic fungi, which can then be used in a method according to the invention. This possibility is opened, among other things, by the high homology between phosphomannomutases from different fungal species (see Fig. 3).
  • the phosphomannomutase from S. cerevisiae is preferably used.
  • polypeptides refers to both short amino acid chains, commonly called peptides, oligopeptides, or oligomers are referred to, as well as to longer amino acid chains, usually called Proteins are called. It includes amino acid chains that are either natural Processes, such as post-translational processing, or by chemical processes, which are state of the art, can be modified. Such modifications can occur in different locations and multiple times in a polypeptide, such as for example on the peptide backbone, on the amino acid side chain, on the amino and / or at the carboxy terminus.
  • acetylations include, for example, acetylations, acylations, ADP ribosylations, amidations, covalent linkages with Flavins, heme fractions, nucleotides or nucleotide derivatives, lipids or lipid derivatives or phosphatidylinositol, cyclizations, disulfide bridging, demethylations, Cystine formation, formylation, gamma carboxylation, glycosylation, Hydroxylations, iodinations, methylations, myristoylations, Oxidations, proteolytic processing, phosphorylation, selenoylation and tRNA-mediated additions of amino acids.
  • polypeptides according to the invention can be in the form of "mature” proteins or as Parts of larger proteins, e.g. as fusion proteins. Furthermore, they can Secreting or "leader” sequences, pro sequences, sequences that are simple Allow purification, such as multiple histidine residues, or additional stabilizing ones Have amino acids.
  • the proteins of the invention can also are present as they are naturally present in their organism of origin which they can be won directly, for example. In the invention Methods can also use active fragments of a phosphomannomutase as long as they are determining the enzymatic activity of the polypeptide or enable their inhibition by a candidate compound.
  • a possible purification process for phosphomannomutase is based on preparative Electrophoresis, FPLC, HPLC (e.g. using gel filtration, Reverse phase or slightly hydrophobic columns), gel filtration, differential Precipitation, ion exchange chromatography or affinity chromatography (cf. Example 1).
  • a quick method to isolate phosphomannomutases from host cells synthesized begins with the expression of a fusion protein, whereby the fusion partner can be easily affinity-cleaned.
  • the fusion partner can be, for example, a GST tag (see example 1).
  • the fusion protein can then be purified on a glutathione-Sepharose column.
  • the fusion partner can by partial proteolytic cleavage, for example on linkers between the Fusion partner and the polypeptide to be purified according to the invention separated become.
  • the linker can be designed to target amino acids such as Arginine and lysine residues include sites for trypsin cleavage define. Standard cloning methods can be used to generate such linkers using oligonucleotides.
  • polypeptides according to the invention can also be used without a fusion partner using Antibodies that bind to the polypeptides are affinity purified.
  • the cells obtained in this way contain the polypeptide according to the invention or the like Purified polypeptide obtained are useful in methods of identifying Modulators or inhibitors of phosphomannomutase to be used.
  • the present invention also relates to the use of polypeptides from fungi which have at least one biological activity of a phosphomannomutase in methods for identifying inhibitors of a polypeptide from fungi with the activity of a phosphomannomutase, it being possible for the phosphomannomutase inhibitors to be used as fungicides.
  • the phosphomannomutase from S. cerevisiae is particularly preferably used in these processes.
  • Fungicidal active ingredients which are found with the help of a phosphomannomutase from a certain fungal species, can also interact with phosphomannomutases from other fungal species, although the interaction with the different phosphomannomutases occurring in these fungi does not always have to be equally strong. This explains, among other things, the selectivity of active substances.
  • the use of the active ingredients found with a specific phosphomannomutase as a fungicide in other fungi can be attributed to the fact that phosphomannomutases from different fungus species are very close and show pronounced homology in larger areas.
  • Figure 3 clearly shows that such homology exists over considerable sequence sections between S. cerevisiae, C. albicans, S. pombe and N. crassa and thus the effect of the substances found with the help of phosphomannomutase from yeast is not on S. cerevisiae remains limited.
  • the present invention therefore also relates to a method for Identify fungicides by testing potential inhibitors or Modulators of the enzymatic activity of phosphomannomutase (candidate compound) in a phosphomannomutase inhibition test.
  • Methods which are suitable for identifying modulators, in particular inhibitors or antagonists, of the polypeptides according to the invention are generally based on determining the activity or the biological functionality of the polypeptide.
  • both whole-cell-based methods in vivo methods
  • methods based on the use of the polypeptide isolated from the cells which can be present in purified or partially purified form or as a crude extract, are also possible.
  • These cell-free in vitro methods can be used just like in vivo methods on a laboratory scale, but preferably also in HTS or UHTS methods.
  • tests can be carried out on fungal cultures in order to test the fungicidal activity of the compounds found.
  • test systems that aim to test compounds and natural extracts are preferably geared towards high throughput numbers in order to maximize the number of substances tested in a given period of time.
  • Test systems that are based on cell-free work require purified or semi-purified protein. They are primarily suitable for a "first" test, which primarily aims to detect a possible influence of a substance on the target protein. Once such a first test has been carried out and one or more compounds, extracts etc. have been found, the effect of such compounds can be examined in a more targeted manner in the laboratory.
  • the inhibition or activation of the polypeptide according to the invention can be checked again in vitro in order to then test the effectiveness of the compound on the target organism, here one or more phytopathogenic fungi.
  • the compound can then optionally be used as a starting point for the further search and development of fungicidal compounds which are based on the original structure, but which are optimized, for example, in terms of effectiveness, toxicity or selectivity.
  • a synthetic reaction mix e.g. products of in vitro transcription
  • a cellular component such as a membrane, a compartment or any other preparation which contains the polypeptides according to the invention
  • an optionally labeled substrate, cofactor or ligand Polypeptides are incubated in the presence and absence of a candidate molecule, which may be an antagonist.
  • the ability of the candidate molecule to inhibit the activity of the polypeptides according to the invention is evident, for example, from a reduced binding of the optionally labeled ligand or from a reduced conversion of the optionally labeled substrate.
  • Molecules that inhibit the biological activity of the polypeptides according to the invention are good antagonists.
  • the detection of the biological activity of the polypeptides according to the invention can be improved by a so-called reporter system.
  • Reporter systems in this Aspects include, but are not limited to, colorimetric or fluorimetric detectable substrates that are converted into a product or a Reporter gene which is based on changes in the activity or expression of the invention Responses to polypeptides or other known binding assays.
  • Another example of a method with which modulators of the invention Polypeptides can be found is a displacement test in which the polypeptides according to the invention and a potential modulator with a molecule known to be according to the invention Polypeptides bind like a natural substrate or ligand or a substrate or ligand mimetic.
  • the invention Polypeptides themselves can be labeled, e.g. fluorimetric or colorimetric, so you can see the number of polypeptides bound to a ligand are or who have participated in an implementation, can determine exactly.
  • binding can be carried out using the optionally labeled substrate, ligand or substrate analogs are tracked. In this way, the effectiveness of the antagonist.
  • Test systems check both inhibitory or suppressive effects of the substances as well as stimulatory effects.
  • the effectiveness of a substance can be checked using concentration-dependent test series. Control approaches without test substances or without enzyme can be used to evaluate the effects.
  • Host cells containing phosphomannomutase-encoding nucleic acids is the development of test systems based on cells for identification of substances that allow the activity of the polypeptides according to the invention modulate.
  • SPA Stentillation Proximity Assay
  • This test system uses the interaction a polypeptide (e.g. yeast phosphomannomutase) with one radiolabeled ligands or substrate.
  • the polypeptide is small Balls ("Microspheres") or beads ("Beads") bound with scintillating Molecules are provided.
  • the test conditions are optimized so that only those particles originating from the ligand lead to a signal from one to the polypeptide of the invention bound ligand.
  • the modulators to be identified are preferably small organic chemical compounds.
  • the compound which has the activity is particularly preferably determined of the polypeptide of the invention specifically inhibited.
  • activity like it is used here refers to the biological activity of the invention Polypeptide.
  • a preferred method takes advantage of the fact that the phosphate group of D-mannose-6-phosphate is acid stable while the phosphate group of D-mannose-1-phosphate is acid labile.
  • the activity or the increase or decrease in the activity of the polypeptide according to the invention can therefore by means of detection of acid cleaved phosphate group from D-mannose-1-phosphate with a phosphate detection reagent be determined.
  • the lower or inhibited Activity of the polypeptide according to the invention based on the photospectrometric Determination of the decrease or the increase in those which can be split off by acid Phosphate group traced by D-mannose-1-P.
  • the concentration of the released Phosphate can then be used with a phosphate detection reagent at an absorption maximum can be determined at 620 nm.
  • the measurement can also be carried out in formats more common for HTS or UHTS assays, e.g. in microtiter plates in which, for example, a total volume of 5 to 50 ⁇ l per batch or per well is placed and the individual components are present in one of the above-mentioned final concentrations ( see Example 2).
  • the compound to be tested potentially inhibiting or activating the activity of the enzyme (candidate molecule), is initially introduced, for example, in a suitable concentration in the above-mentioned test buffer containing D-mannose-1-phosphate.
  • the polypeptide according to the invention is then added in the above-mentioned test buffer containing NADP + , glucose and the auxiliary enzymes phosphoglucose isomerase and glucose-6-phosphate dehydrogenase necessary for the coupled assay, and the reaction is started therewith.
  • the mixture is then incubated, for example, for up to 2 or 3 hours at a suitable temperature and the increase in fluorescence is measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
  • a further measurement is carried out in a corresponding approach, but without addition of a candidate molecule and without adding a polypeptide according to the invention (Negative control).
  • Another measurement takes place in the absence of one Candidate molecule, but in the presence of the polypeptide according to the invention (Positive control).
  • Negative and positive control result in the comparison values approaches in the presence of a candidate molecule.
  • the pI50 value given there is the negative decimal logarithm of the so mentioned IC50 value, which indicates the molar concentration of a substance that is used for 50% inhibition of the enzyme results.
  • a pI50 value of 8 corresponds, for example, to a half-maximum inhibition of the enzyme at a concentration of 10 nM.
  • the inhibitors of a phosphomannomutase according to the invention identified with the aid of a method according to the invention are suitable for damaging or killing fungi.
  • the cavities of microtiter plates testing active ingredient are pipetted. After the solvent evaporated medium is added to each cavity.
  • the medium is previously with a suitable concentration of spores or mycelium of the fungus to be tested.
  • the resulting concentrations of the active ingredient are e.g. 0.1, 1, 10 and 100 ppm.
  • the plates are then placed on a shaker at a temperature of 22 ° C incubated until sufficient growth is detectable in the untreated control is.
  • the evaluation is then carried out photometrically at a wavelength of 620 nm. From the measurement data of the different concentrations, the dose of active substance can be determined, which leads to a 50% inhibition of fungal growth compared to the untreated control (ED 50 ).
  • Another method of testing the fungicidal activity is based on the to test the protective effect of the compound to be tested on a plant. To the plant is sprayed with a solution of the active ingredient to be tested and then inoculated with a solution of fungal spores and for several days watched away. It can then be compared to a plant that is not pre-made an active substance solution has been treated, it can be determined whether the substance to be tested Active ingredient prevents or reduces the infestation or spread of the fungus.
  • the active ingredient concentration can also be determined here, which leads to a 50% inhibition of fungal growth compared to the untreated control (ED 50 ).
  • Efficiency can be used, where 0% stands for an efficiency that corresponds to that of the control, while an efficiency of 100% means that no infestation is observed.
  • Table II shows the results of such a test as an ED 50 value and as an efficiency (WG) for a compound found in a process according to the invention (Table I, Example 2).
  • organism ED 50 [ppm] Flat share [%] Phytophtora infectans 500 33
  • the present invention now enables based on what is disclosed herein Knowledge regardless of the specific compounds disclosed herein that only to clarify or demonstrate the principle of the invention - new ones Identify or optimize fungicidal active ingredients. So now you can any compounds with regard to their effect on the so far not as a target enzyme known for fungicides, phosphomannomutase both tested, fungicidal Connections identified and optimized in an efficient way. To can e.g. uses the methods disclosed in the present invention become.
  • the present invention therefore also relates to the use of Modulators of phosphomannomutase from fungi, preferably from plant pathogens Fungi as fungicides.
  • the present invention also relates to fungicides which are applied using a inventive method can be identified.
  • Active substances that are identified with the aid of a method according to the invention and / or inhibit the enzymatic activity of the phosphomannomutase therefore in certain concentrations and application rates as fungicides be used. If necessary, they can also be used as intermediate and intermediate products for the optimization of fungicidal agents and for synthesis use other active ingredients.
  • plants and parts of plants can be used with these active ingredients be treated.
  • Plants include all plants and plant populations understood how desired and unwanted wild plants or crops (including naturally occurring crops). crops can be plants by conventional breeding and optimization methods or by biotechnological and genetic engineering methods or Combinations of these methods can be obtained, including the transgenic plants and including those that can be protected by plant breeders' rights or non-protectable plant varieties.
  • Under plant parts all above-ground and underground parts and organs of plants, such as sprout, leaf, flower and Root can be understood, examples being leaves, needles, stems, stems, Flowers, fruiting bodies, fruits and seeds as well as roots, tubers and rhizomes be performed.
  • the plant parts also include crops and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, Offshoots and seeds.
  • the treatment according to the invention of the plants and parts of plants with the said Active substances take place directly or by influencing their environment, living space or storage room according to the usual treatment methods, e.g. by diving, Spraying, vaporizing, atomizing, scattering, spreading and in the case of propagation material, especially in the case of seeds, further by single or multi-layer coating.
  • the active ingredients can depend on their respective physical and / or chemical properties are converted into the usual formulations, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, Aerosols, very fine encapsulations in polymeric substances and in coating materials for seeds, as well as ULV cold and warm fog formulations.
  • formulations are made in a known manner, e.g. by mixing of the active substances with extenders, i.e. liquid solvents, under pressure liquefied gases and / or solid carriers, optionally using of surface-active agents, i.e. emulsifiers and / or dispersants and / or foam-generating agents.
  • extenders i.e. liquid solvents
  • surface-active agents i.e. emulsifiers and / or dispersants and / or foam-generating agents.
  • surface-active agents i.e. emulsifiers and / or dispersants and / or foam-generating agents.
  • organic solvents are also used as auxiliary solvents become.
  • aromatics such as xylene, toluene or alkylnaphthalenes
  • chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chlorethylenes or methylene chloride
  • aliphatic hydrocarbons such as cyclohexane or paraffins, e.g. Petroleum fractions
  • Alcohols such as butanol or glycol
  • ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone
  • strongly polar Solvents such as dimethylformamide and dimethyl sulfoxide, and water.
  • Liquefied gaseous extenders or carriers are such liquids meant which are gaseous at normal temperature and pressure, e.g. Aerosol propellants, such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.
  • Aerosol propellants such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.
  • Solid carriers are possible: e.g. natural rock flour, such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth and synthetic rock powder, such as highly disperse silica, Alumina and silicates. Coming as solid carriers for granules in question: e.g.
  • emulsifier and / or foam generator Funds are possible: e.g. non-ionic and anionic emulsifiers, such as polyoxyethylene fatty acid esters, Polyoxyethylene fatty alcohol ethers, e.g. alkylaryl, Alkyl sulfonates, alkyl sulfates, aryl sulfonates and protein hydrolyzates.
  • Dispersants are possible: e.g. Lignin sulfite liquor and methyl cellulose.
  • adhesives such as carboxymethyl cellulose, natural and synthetic powdery, granular or latex-shaped polymers are used, such as gum arabic, polyvinyl alcohol, polyvinyl acetate, and natural phospholipids, such as cephalins and lecithins, and synthetic phospholipids.
  • Other additives can be mineral and vegetable oils.
  • Dyes such as inorganic pigments, e.g. Iron oxide, titanium oxide, ferrocyan blue and organic dyes such as alizarin, azo and metal phthalocyanine dyes and trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, Molybdenum and zinc can be used.
  • inorganic pigments e.g. Iron oxide, titanium oxide, ferrocyan blue and organic dyes such as alizarin, azo and metal phthalocyanine dyes and trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, Molybdenum and zinc
  • trace nutrients such as salts of iron, manganese, boron, copper, cobalt, Molybdenum and zinc
  • the active substances can be used as such, in the form of their formulations or in the formulations prepared from them
  • Application forms such as ready-to-use solutions, suspensions, wettable powders, Pastes, soluble powders, dusts and granules can be used.
  • the Application takes place in the usual way, e.g. by pouring, spraying, spraying, Scattering, dusting, foaming, brushing etc. It is also possible to apply the active ingredients according to the ultra-low-volume process or the Active ingredient preparation or to inject the active ingredient yourself into the soil. It can also the seeds of the plants are treated.
  • the application rates can be varied within a wide range depending on the type of application.
  • the amounts of active ingredient are in the Generally between 0.1 and 10,000 g / ha, preferably between 10 and 1,000 g / ha.
  • the active compound application rates are in the Generally between 0.001 and 50 g per kilogram of seed, preferably between 0.01 and 10 g per kilogram of seed.
  • the application rates of active ingredient generally between 0.1 and 10,000 g / ha, preferably between 1 and 5,000 g / ha.
  • the ORF was amplified from genomic DNA from Saccharomyces cerevisiae via gene-specific primers.
  • the corresponding DNA an amplicon of 765 bp in length, was intercloned into the vector pGEX-4T-1 from Pharmacia Biotech and then via the interfaces Bam HI and Xho I introduced through the primers into the vector pGEX cut with interfaces Bam HI and Xho I -4T-1 (Pharmacia Biotech) cloned.
  • the resulting plasmid pSec53 contains the complete coding sequence of sec53 in an N-terminal fusion with the GST tag, which is part of the vector.
  • the Sec53 fusion protein has a calculated mass of 53 kD.
  • the plasmid pSec53 was transformed into E. coli BL21 (DE3) in such a way that the transformation mixture served directly as a preculture in 50 ml of selection medium. These cells were incubated overnight at 37 ° C. and then diluted 1: 100 in selection medium (LB medium with 100 ⁇ g / ml ampicillin). At an OD 600nm of 0.8 - 1.0 the temperature of the cells was reduced to 18 ° C and induced with 1mM IPTG (final concentration). The cells were harvested after 24 hours. The cell pellets can be stored for several months at -80 ° C without loss of activity.
  • the digestion was carried out by sonification in lysis buffer (200 mM KCl, 10 mM imidazole, 50 mM Tris-HCl, pH 8, 15% glycerol). The cleaning was carried out according to the manufacturer's standard protocol for glutathione-Sepharose columns. The purified protein was then mixed with glycerol in buffer (50 mM Tris-HCl pH 8, 5 mM glutathione, 15% glycerol) and stored at -80 ° C. About 3.5 mg of soluble protein could be isolated from 250 ml of culture medium, which could be used in methods for identifying phosphomannomutase modulators.
  • 384-well microtiter plates were used to identify modulators of phosphomannomutase used by Greiner.
  • the negative control was pipetted into the first column. This was composed of 5 ⁇ l test buffer (2 mM MgCl 2 , 100 mM Tris / HCl, pH 7, 1 mM EDTA) with 5% DMSO, 20 ⁇ l test buffer (2 mM MgCl 2 , 100 mM Tris / HCl, pH 7, 1 mM EDTA) and 25 ⁇ l Mix 1 (100 mM Tris / HCl pH 7, 2 mM MgCl 2 , 1 mM EDTA, 1 mM NADP + , 0.2 mM D-glucose-1,6-diphosphate, 0.0125 U Phosphoglucose isomerase, 0.0125 U phosphomannose isomerase, 0.05 U glucose-6-phosphate dehydrogenase, 0.05 ⁇ g phosphomannomutase).
  • the positive control was pipetted into the second column. This consisted of 5 ⁇ l test buffer with 5% DMSO, 20 ⁇ l Mix 2 (1 mM D-mannose-1-phosphate, 2 mM MgCl 2 , 100 mM Tris / HCl, pH 7, 1 mM EDTA) and 25 ⁇ l Mix 1 (100 mM Tris / HCl pH 7, 2 mM MgCl 2 , 1 mM EDTA, 1 mM NADP + , 0.2 mM D-glucose-1,6-diphosphate, 0.0125 U phosphoglucose isomerase, 0.0125 U phosphomannose isomerase , 0.05 U glucose-6-phosphate dehydrogenase, 0.05 ⁇ g phosphomannomutase).
  • a test substance at a concentration of 2 ⁇ M in DMSO was placed in the remaining columns, the test buffer being used to dilute the substance to a volume of 5 ⁇ l.
  • 20 ⁇ l Mix 2 (1 mM D-mannose-1-phosphate, 2 mM MgCl 2 , 100 mM Tris / HCl, pH 7, 1 mM EDTA), 25 ⁇ l Mix 1 (100 mM Tris / HCl pH 7, 2 mM MgCl 2 , 1 mM EDTA, 1 mM NADP + , 0.2 mM D-glucose-1,6-diphosphate, 0.0125 U phosphoglucose isomerase, 0.0125 U phosphomannose isomerase, 0.05 U Glucose-6-phosphate dehydrogenase, 0.05 ug expressed and purified phosphomannomutase) added. This was followed by incubation at room temperature for 20 minutes.
  • the NADPH formed during the reaction was measured by determination absolute fluorescence in a Tecan Ultra suitable for MTP Fluorescence spectrometer.
  • a methanolic solution of the was placed in the wells of microtiter plates of an active ingredient identified according to the invention (Tab. I, Ex. 2), mixed with an emulsifier, pipetted. After the solvent evaporated 200 ⁇ l of potato dextrose medium were added to each cavity. The medium was previously treated with suitable concentrations of spores or mycelia testing mushroom (see Table II).
  • the resulting concentrations of the active ingredient were 0.1, 1, 10 and 100 ppm.
  • the resulting concentration of the emulsifier was 300 ppm.
  • the plates were then incubated on a shaker at a temperature of 22 ° C. until sufficient growth was found in the untreated control.
  • the evaluation was carried out photometrically at a wavelength of 620 nm.
  • the dose of active ingredient which leads to a 50% inhibition of fungal growth compared to the untreated control (ED 50 ) is calculated from the measurement data of the different concentrations.
  • Compound 2 from Table I already showed a clearly fungicidal activity at the application rate given in Table II.

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DE2002127001 DE10227001A1 (de) 2002-06-18 2002-06-18 Verfahren zum Identifizieren von fungizid wirksamen Verbindungen

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Citations (2)

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Publication number Priority date Publication date Assignee Title
GB1434040A (en) * 1973-08-06 1976-04-28 Ici Ltd Process for combating fungi and bacteria
WO1999047698A1 (fr) * 1998-03-19 1999-09-23 Mcgill University Criblage au moyen de genes codant pour mannosyltransferase de s. cerivisiae

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1434040A (en) * 1973-08-06 1976-04-28 Ici Ltd Process for combating fungi and bacteria
WO1999047698A1 (fr) * 1998-03-19 1999-09-23 Mcgill University Criblage au moyen de genes codant pour mannosyltransferase de s. cerivisiae

Non-Patent Citations (6)

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Title
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1976, MURATA T: "PURIFICATION AND SOME PROPERTIES OF PHOSPHO MANNO MUTASE FROM CORMS OF AMORPHOPHALLUS-KONJAC", XP002258028, Database accession no. PREV197763071351 *
GLASER L., ET AL.: "Preparation and Properties of Phosphomannomutase from Baker's Yeast", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 33, no. 2, 1959, pages 522 - 6, XP008023380, ISSN: 0006-3002 *
MARTIN A ET AL: "6R- and 6S-6C-methylmannose from D-mannuronolactone. Inhibition of phosphoglucomutase and phosphomannomutase: Agents for the study of the primary metabolism of mannose", TETRAHEDRON ASYMMETRY 29 JAN 1999 UNITED KINGDOM, vol. 10, no. 2, 29 January 1999 (1999-01-29), pages 355 - 366, XP004158856, ISSN: 0957-4166 *
PLANT AND CELL PHYSIOLOGY, vol. 17, no. 6, 1976, pages 1099 - 1109, ISSN: 0032-0781 *
WELLS TIMOTHY N C ET AL: "Mechanism of irreversible inactivation of phosphomannose isomerases by silver ions and flamazine", BIOCHEMISTRY, vol. 34, no. 24, 1995, pages 7896 - 7903, XP002258071, ISSN: 0006-2960 *
WILLS ELIZABETH A ET AL: "Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity", MOLECULAR MICROBIOLOGY, vol. 40, no. 3, May 2001 (2001-05-01), pages 610 - 620, XP002258026, ISSN: 0950-382X *

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JP2005529600A (ja) 2005-10-06
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AU2003242661A1 (en) 2003-12-31

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